title: Transgenically mediated shRNAs targeting conserved regions of foot-and-mouth disease virus provide heritable resistance in porcine cell lines and suckling mice.
abstract: Foot-and-mouth disease virus (FMDV) is responsible for substantial economic losses in livestock breeding each year, and the development of new strategies is needed to overcome the limitations of existing vaccines and antiviral drugs. In this study, we evaluated the antiviral potential of transgenic porcine cells and suckling mice that simultaneously expressed two short-hairpin RNAs (shRNAs) targeting the conserved regions of the viral polymerase protein 3D and the non-structural protein 2B. First, two recombinant shRNA-expressing plasmids, PB-EN3D2B and PB-N3D2B, were constructed and the efficiency of the constructs for suppressing an artificial target was demonstrated in BHK-21 cells. We then integrated PB-EN3D2B into the genome of the porcine cell line IBRS-2 using the piggyBac transposon system, and stable monoclonal transgenic cell lines (MTCL) were selected. Of the 6 MTCL that were used in the antiviral assay, 3 exhibited significant resistance with suppressing ratios of more than 94% at 48 hours post-challenge (hpc) to both serotype O and serotype Asia 1 FMDV. MTCL IB-3D2B-6 displayed the strongest antiviral activity, which resulted in 100% inhibition of FMDV replication until 72 hpc. Moreover, the shRNA-expressing fragment of PB-N3D2B was integrated into the mouse genome by DNA microinjection to produce transgenic mice. When challenged with serotype O FMDV, the offspring of the transgenic mouse lines N3D2B-18 and N3D2B-81 exhibited higher survival rates of 19% to 27% relative to their non-transgenic littermates. The results suggest that these heritable shRNAs were able to suppress FMDV replication in the transgenic cell lines and suckling mice.
authors.forename: Ye
authors.initials: Y
authors.lastname: Jiao
authors.forename: Xiuli
authors.initials: X
authors.lastname: Gong
authors.forename: Junzheng
authors.initials: J
authors.lastname: Du
authors.forename: Mingqiu
authors.initials: M
authors.lastname: Liu
authors.forename: Xinbing
authors.initials: X
authors.lastname: Guo
authors.forename: Linlin
authors.initials: L
authors.lastname: Chen
authors.forename: Weinan
authors.initials: W
authors.lastname: Miao
authors.forename: Tao
authors.initials: T
authors.lastname: Jin
authors.forename: Huiyun
authors.initials: H
authors.lastname: Chang
authors.forename: Yitao
authors.initials: Y
authors.lastname: Zeng
authors.forename: Zhaoxin
authors.initials: Z
authors.lastname: Zheng
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D000833:Animals, Suckling; D002460:Cell Line; D017124:Conserved Sequence; D060467:Disease Resistance; D005536:Foot-and-Mouth Disease / Q000235:genetics* / Q000517:prevention & control* / Q000821:virology; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics* / Q000276:immunology; D015967:Gene Expression Regulation, Viral; D051379:Mice; D010957:Plasmids / Q000235:genetics / Q000276:immunology; D016133:Polymerase Chain Reaction; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D013552:Swine; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology
chemical_list: D034741:RNA, Small Interfering; D014764:Viral Proteins